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. 2021 Jun 18;10:e61407. doi: 10.7554/eLife.61407

Figure 3. Proximity ligation assay (PLA) analysis of cavin3 and BRCA1 interaction in MCF7 cells.

(A–E) Immunofluorescence microscopy in combination with PLA for protein-protein interactions (red dots) within single cells of stably expressing (A) MCF7-GFP, (B) MCF7-cavin1-GFP, (C) MCF7-cavin2-GFP, (D) MCF7-cavin3-GFP, and (E) MCF7-CAV1-GFP using monoclonal GFP and polyclonal BRCA1 antibodies. DNA was counterstained with DAPI (blue). Scale bars represent 10 μm. (F). Number of red dots/PLA signals in 40–50 cells for each MCF7-GFP-expressing cell line was quantified from three independent experiments using a nested ANOVA. Each biological replicate is color-coded, and the mean ± SEM is presented as a black bar. **p<0.05, **p<0.01.

Figure 3.

Figure 3—figure supplement 1. Proximity ligation assay (PLA) demonstrates cavin3 and BRCA1 interaction in MCF7 cells.

Figure 3—figure supplement 1.

(A) Immunofluorescence microscopy in combination with PLA to detect and visualize protein-protein interactions (red dots) within single cells of stably expressing MCF7-GFP and (B) MCF7-cavin3-GFP cells using monoclonal BRCA1 (MS 110) and polyclonal GFP antibodies. DNA was counterstained with DAPI (blue). Scale bars represent 10 μm. (C) Number of red dots/PLA signals in 40–50 cells for each MCF7-GFP-expressing cell line quantified from three independent experiments using a nested ANOVA. Each biological replicate is color-coded with the mean ± SEM presented as a black bar. **p<0.01.
Figure 3—figure supplement 2. Proximity ligation assay (PLA) controls.

Figure 3—figure supplement 2.

(A) Fluorescence microscopy analysis of PLA signals generated in MCF7 cells transfected with cavin3-GFP using mouse GFP and rabbit BRCA1 antibodies from Figure 3D, (B) GFP antibody alone, (C) BRCA1 antibody alone, (D) the absence of PLA probes, or (E) primary antibody controls. Representative images are from at least two independent experiments as shown.